SERS (Surface Enhanced Raman Scattering) nanoprobe for quantitatively detecting pesticide residues in water and detection method

Abstract

The invention belongs to but not limited to the technical field of pesticide residue detection, and discloses an SERS (Surface Enhanced Raman Scattering) nanoprobe for quantitatively detecting pesticide residues in water and a detection method, TMV (Tetramethylbenzidine) is adopted as a biological template, and a TMV-Pt seed is formed through rapid nucleation of Pt atoms on the outer surface of the TMV; sodium polystyrenesulfonate and ascorbic acid are used for finely regulating and controlling the growth direction of Pt seeds on the surface of the TMV; pt is promoted to form a cluster with a dendritic structure in a limited space; the diameter of the dendritic metal cluster is adjusted by controlling the reaction time, and finally TMV-DPtNCs are formed on the surface of the TMV; preparing a TMV-DPtNCs (at) rGO composite material by using the reduced graphene oxide; based on TMV-DPtNCs (at) rGO, pesticide residues in water are detected by utilizing an SERS technology. According to the invention, the lowest detection limit on the three pesticides, namely the dalarin, the carbaryl and the triazophos, can reach 10 <-7 > M; the detection on a real water sample is met.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical field of pesticide residue detection, and in particular relates to a SERS nanoprobe and a detection method for quantitatively detecting pesticide residues in water. Background Art

[0002] The detection of pesticide residues in water is a key issue in the field of environmental monitoring and food safety. Although traditional detection methods such as chromatography, mass spectrometry, electrochemistry and biosensor technology have high accuracy, they often have limitations such as complex operation, expensive equipment, inconvenience in carrying, and difficulty in real-time monitoring. In contrast, surface enhanced Raman spectroscopy (SERS) technology has shown broad application potential due to its advantages of being non-destructive, rapid, sensitive and reliable, especially in water sample analysis without interference.

[0003] The key to SERS technology lies in the construction of enhanced substrates, and high-density and uniformly distributed "hotspot" structures are the core of improving detection sensitivity. However, existing artificial synthesis methods, such as chemical reduction, photolithography, and template methods, are difficult to form ideal uniform and dense "hotspot" structures, limiting the practical application of SERS. In addition, these methods usually involve complex chemical processes and harmful reagents, which do not meet the requirements of green environmental protection. The preparation of high-quality SERS substrates is still a global problem, which restricts the practical application of SERS technology.

[0004] After millions of years of evolution, nature has formed highly uniform and complex biological material surface structures at the nanometer and submicrometer scales, such as plant leaves, petals, and insect wings. These materials not only provide inspiration for the preparation of high-performance SERS substrates, but also have good biocompatibility and degradability, which can effectively reduce environmental impact. At present, some studies have used these natural materials to construct SERS substrates, but the exploration of using plant viruses as templates is still rare.

[0005] In view of the above analysis, the problems existing in the prior art are: it is difficult to prepare a SERS substrate with high uniformity and dense “hot spots” using existing artificial synthesis methods. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for preparing a novel nanoprobe. Based on the nanoprobe, a method for detecting pesticide residues in water using surface enhanced Raman scattering (SERS) technology is established.

[0007] The present invention is achieved by providing a SERS nanoprobe for quantitative detection of pesticide residues in water. The preparation method of the SERS nanoprobe for quantitative detection of pesticide residues in water comprises the following steps:

[0008] Step 1: Using tobacco mosaic virus (TMV) as a biological template, using the widely distributed arginine residues on its surface as inducing groups, reducing metal ions platinum (Pt) (single or multiple metals such as gold, silver, palladium, platinum, copper, etc. can be selected) to promote the rapid nucleation of Pt atoms on the outer surface of TMV to form TMV-Pt seeds;

[0009] Step 2: Use sodium polystyrene sulfonate (PSS) and ascorbic acid (AA) to finely control the growth direction of Pt seeds on the TMV surface, and promote Pt to form dendritic clusters in a limited space (2.5 nm in the axial direction and 3.5 nm in the radial direction).

[0010] Step 3: The diameter of the dendritic metal clusters was adjusted by controlling the reaction time, and finally Pt-covered one-dimensional nanowires (TMV-DPtNCs) were formed on the TMV surface.

[0011] Step 4: Prepare TMV-DPtNCs@rGO composite material using reduced graphene oxide (rGO); the addition of rGO can quench the fluorescence background of TMV-DPtNCs and provide a chemical enhancement (CM) effect for surface enhanced Raman scattering (SERS).

[0012] Further, the purification method of tobacco mosaic virus (TMV) comprises:

[0013] Take about 100g of tobacco leaves (frozen and stored in a -80℃ refrigerator), crush them, add 300mL potassium phosphate buffer solution (100mmol / L, pH 7.8) and 1mL β-mercaptoethanol, crush them, and filter out the residue with double-layer gauze; the filtrate is centrifuged at 10580r / min for 20min at 4℃, and the supernatant is taken; an equal volume of chloroform-n-butanol mixed solvent (v:v=1:1) is added, and the mixture is stirred in an ice bath for 30min; the mixture is centrifuged at 6045r / min for 10min at 4℃, and the upper layer (water layer) is taken; PEG 8000 is added to a final concentration of 10% (w / V); sodium chloride is added to a final concentration of 200mmol / L, and the mixture is stirred in an ice bath for 30min; the mixture is centrifuged at 14106rpm for 30min at 4℃, the supernatant is poured out, and 50mL potassium phosphate buffer solution (10mmol / L, pH 7.8) Redissolve, centrifuge at 9500 r / min for 15 min, take the supernatant; centrifuge at 45000 r / min for 2.5 h at 4°C, and redissolve the precipitate with 10 mL potassium phosphate buffer solution (10 mmol / L, pH 7.8); centrifuge the resolvent at 9500 r / min for 15 min at 4°C, and store at 4°C; the TMV concentration is semi-quantitatively determined by measuring the ultraviolet absorption at 260 nm, and the extinction coefficient is 3.1 cm 2 / g.

[0014] Further, the synthesis method of TMV-Pt seeds:

[0015] 15 mg of sodium polystyrene sulfonate (PSS) was added to a TMV aqueous solution (15 mL, 6 mg) and stirred to dissolve; 5 mL of K2PtCl4 solution (20 mM) was added dropwise and stirred at room temperature for 30 min. The arginine groups evenly distributed on the surface of TMV were used to adsorb [PtCl4] 2- ; Then, a freshly prepared NaBH4 solution (20 mL, 5 mM) was quickly poured in at once, and the mixture was stirred rapidly for 2 h to generate Pt nanoparticles with a particle size of about 2 nm on the surface of TMV;

[0016] After the reaction is completed, the reaction solution is transferred to a centrifuge tube, cushioned with 20% glycerol, and centrifuged at 9400g for 30 minutes; carefully remove the supernatant, add ultrapure water to the precipitate to resuspend, centrifuge at 6000g for 20 minutes, and wash the precipitate; after washing three times, add ultrapure water to the precipitate to resuspend, centrifuge at 500g for 10 minutes, collect the supernatant and store it at 4°C, which is the TMV-Pt seed.

[0017] Further, the synthesis method of continuous multichannel Pt nanowires (TMV-DPtNCs) with dendritic structure:

[0018] Weigh 240 mg of polyvinylpyrrolidone (PVP) (K-30), dissolve it in 32 mL of water, add TMV-Pt seeds to make the final concentration 10 μg / mL; add K2PtCl4 solution (4 mL, 10 mM) under stirring; finally add freshly prepared ascorbic acid (AA) solution (4 mL, 100 mM), stir and react at room temperature overnight;

[0019] After the reaction, the reaction solution was transferred to a centrifuge tube, centrifuged at 6500g for 20 min, and the supernatant was discarded; ultrapure water was added to the precipitate to resuspend it, and the precipitate was washed by centrifugation; after washing three times, the precipitate was dispersed in ultrapure water and stored at 4°C to obtain TMV-DPtNCs.

[0020] Further, the preparation method of TMV-DPtNCs@rGO composite stock solution:

[0021] 100 μL of TMV-DPtNCs and a certain amount of rGO were mixed to ensure that the final concentration of rGO was 0.005 g / L and stored in a refrigerator at -4 °C.

[0022] Another object of the present invention is to provide a detection method of a SERS nanoprobe for quantitative detection of pesticide residues in water, comprising the following steps:

[0023] Step 1: Using the fluorescent probe molecule rhodamine 6G (R6G) in combination with Raman spectroscopy, the Raman enhancement effect of TMV-DPtNCs@rGO was evaluated and its enhancement factor (EF) was calculated;

[0024] Step 2, evaluate the SERS enhancement effect of TMV-DPtNCs@rGO on three commonly used pesticides (diflunisal, carbaryl, and triazophos); establish the linear relationship between the characteristic peak intensity and concentration of these pesticides by the least squares method for quantitative detection;

[0025] Step 3, the standard addition method was used to evaluate the detection ability of TMV-DPtNCs@rGO for three pesticide residues in actual lake water samples.

[0026] Furthermore, the Raman performance of the TMV-DPtNCs@rGO composite for the fluorescent probe (R6G) was tested:

[0027] Take 2 μL of TMV-DPtNCs@rGO composite and 2 μL of different concentrations (10 -4 M to 10 -9 The mixture was mixed with an aqueous solution of R6G (M), placed on a hydrophilic silicon wafer and dried, and then detected using a Raman spectrometer to calculate the EF value, RSD value, etc.

[0028] Furthermore, the Raman performance of TMV-DPtNCs@rGO composite for three pesticides (triazophos, cypermethrin and carbaryl) was tested:

[0029] Take 2 μL of TMV-DPtNCs@rGO composite and 2 μL of different concentrations (10 -4 M to 10 -7 The mixture of pesticides was placed on a hydrophilic silicon wafer and dried before being detected using a Raman spectrometer. The linear relationship between the characteristic peak intensity and concentration of the pesticides was established using the least squares method for quantitative detection.

[0030] Furthermore, the TMV-DPtNCs@rGO composite was used to detect three pesticides in real environmental water samples:

[0031] Take natural lake water and add three pesticides of different concentrations (5.00×10 -4 M to 5.00×10 -7 M), and then filtered using a 0.22 μm nylon filter membrane; then 2 μL of water sample was mixed with 2 μL of TMV-DPtNCs@rGO complex, placed on a hydrophilic silicon wafer for drying and then subjected to Raman detection.

[0032] Furthermore, the conditions for the Raman spectrometer to perform detection are: 532 nm, 1% energy, 5 seconds integration time, and integration times: 1.

[0033] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0034] First, the present invention addresses the problem that it is difficult to prepare a highly uniform SERS substrate with dense "hot spots" using existing artificial synthesis methods, and proposes to use tobacco mosaic virus (TMV) as a biological template for SERS enhanced substrates. TMV has a hollow rod-like structure, highly uniform nanoscale dimensions (300nm long, 18nm outer diameter, 4nm inner diameter), and abundant functional groups. It can construct highly uniform one-dimensional metal nanowires by depositing metal ions to form a dense SERS "hot spot" structure. TMV is non-toxic and degradable to humans, and has the advantage of being green and environmentally friendly. It provides an innovative solution to improve the sensitivity and practicality of SERS technology in pesticide residue detection, and helps promote its application in environmental monitoring and food safety.

[0035] The present invention uses tobacco mosaic virus (TMV) as a biological template for SERS enhancement substrate, and constructs continuous multi-channel Pt nanowires (TMV-DPtNCs) with a dendritic structure through a seed growth method (single or multiple metals such as gold, silver, palladium, platinum, copper, etc. can be selected, and (Pt) is used as an example in the present invention). The dendritic structure of TMV-DPtNCs can provide uniform and dense "hot spots" to enhance the Raman signal. The TMV-DPtNCs@rGO complex formed by the combination of TMV-DPtNCs and rGO can not only eliminate the fluorescence background of the biological template, but also further enhance the Raman effect through the CM effect. Combined with a confocal Raman spectrometer, the minimum detection concentration of the complex for the fluorescent probe R6G can reach 10 -9 M, EF value is 1.18×10 6 The RSD was 4.19%. The minimum detection limit of three pesticides (diflunisal, carbaryl, and triazophos) could reach 10 -7 M. In the test of real lake water, when the pesticide concentration is as low as The recoveries of the three pesticide residues were between 80% and 120%, which can meet the requirements for the detection of real water samples.

[0036] Second, as auxiliary evidence of the inventiveness of the present invention, it is also reflected in the following important aspects:

[0037] (1) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0038] There is no report on using TMV as a template to prepare SERS substrates at home and abroad.

[0039] (2) The technical solution of the present invention solves a technical problem that people have been eager to solve but have never been able to solve successfully:

[0040] In view of the difficulty that the existing synthesis methods are difficult to prepare a SERS substrate with high uniformity and dense "hot spots", the present invention proposes to use tobacco mosaic virus (TMV) as a biological template for SERS enhanced substrates, and construct continuous multi-channel Pt nanowires (TMV-DPtNCs) with a dendritic structure by a seed growth method. TMV-DPtNCs are highly uniform with an average particle size of 62.1nm. As a Raman substrate, they can provide a large number of SERS "hot spots" to enhance the signal and sensitivity. However, this highly uniform and densely "hot spot" SERS substrate cannot be prepared by artificial means. At the same time, in order to further improve its Raman signal, the present invention introduces reduced graphene oxide, which can effectively eliminate the fluorescence background of the biological template itself and provide a chemical enhancement (CM) effect.

[0041] Third, the technical solution of the present invention solves multiple key technical problems in the prior art in industrial applications and has achieved significant technical progress.

[0042] 1. Technical problems solved:

[0043] The complexity and insufficiency of nanoprobe preparation: In traditional methods, the preparation process of nanoprobes is often complicated, and the stability and consistency of the prepared nanomaterials in use are poor. The present invention uses TMV as a biological template and successfully prepares dendritic continuous multi-pass platinum nanowires (TMV-DPtNCs) by seed growth method, which greatly simplifies the preparation process and significantly improves the stability and consistency of nanoprobes.

[0044] Insufficient SERS detection sensitivity: In the existing SERS (surface enhanced Raman spectroscopy) detection technology, the generation and distribution of hot spots are uneven, resulting in low detection sensitivity. The present invention provides a uniform and dense hot spot distribution by preparing TMV-DPtNCs with a dendritic structure, and introduces reduced graphene oxide to eliminate the fluorescence background and improve the chemical enhancement effect, which greatly enhances the Raman signal and improves the application potential of SERS in food safety and environmental monitoring.

[0045] Purification and application limitations of biological templates: In the prior art, the purification process of biological templates is complicated and inefficient, which affects its wide application in the preparation of nanomaterials. The present invention optimizes the purification process of TMV, so that high-purity TMV can be obtained in a relatively short time, and is successfully applied in the preparation of high-efficiency nanomaterials, thus enhancing the industrial application potential of biological templates.

[0046] 2. Significant technological progress achieved:

[0047] Innovative and efficient preparation process: This invention combines TMV bio-template with seed growth method for the first time and successfully prepares nanoprobes with unique dendritic structure. This innovative preparation process not only simplifies the operation process, but also significantly improves the consistency and stability of the product, providing strong support for large-scale industrial production.

[0048] Significant improvement in detection performance: By optimizing the structural design of the nanoprobe, the present invention significantly enhances the sensitivity and specificity of the SERS detection technology. In the detection of pesticide residues in water, the nanoprobe of the present invention can achieve rapid and accurate detection at low concentrations, significantly improving the feasibility and practicality of the SERS technology in practical applications.

[0049] Broad application prospects: The nanoprobe prepared by the present invention not only performs well in the detection of pesticide residues in water, but can also be promoted and applied to other fields such as environmental monitoring, food safety testing and biomedical testing, and has broad market application prospects and economic benefits.

[0050] In summary, the present invention has achieved remarkable technical progress in the preparation of nanoprobes and their application in SERS detection, successfully solved many difficult problems in the prior art, and provided a new technical path for the development of related industries.

[0051] Fourth, the present invention proposes a method for quantitative detection of pesticide residues in water based on SERS (surface enhanced Raman scattering) nanoprobes, using tobacco mosaic virus (TMV) as a biological template, modifying platinum nanowires on its surface, and combining reduced graphene oxide (rGO) to achieve high sensitivity and high selectivity detection of pesticide residues in water. The following are the key parameters of the present invention and the technical problems it solves, as well as significant technical advances.

[0052] 1. Key parameters

[0053] TMV template: As a biological template, TMV contains abundant arginine residues on its surface, which can effectively induce the nucleation and growth of platinum (Pt) nanoparticles. The length and diameter of TMV are about 100nm and 18nm, which provides a good template support for the subsequent formation of platinum nanowires.

[0054] Pt nanoparticle and nanowire size: The diameter of the Pt nanoparticles formed on the TMV surface is about 2 nm. By finely controlling the reaction conditions, continuous Pt nanowires with 2.5 nm and 3.5 nm dendritic structures are formed in the axial and radial directions, further improving the SERS enhancement effect.

[0055] Addition of rGO: Reduced graphene oxide (rGO) not only effectively quenched the fluorescence background of the nanowires, but also provided a chemical enhancement effect for the SERS probe. The final concentration of rGO was controlled at 0.005 g / L, ensuring the sensitivity and signal clarity of the probe.

[0056] 2. Existing technical problems solved

[0057] Insufficient SERS sensitivity: Traditional SERS nanoprobes have low sensitivity for detecting pesticide residues in water and cannot accurately detect trace pesticide residues. The present invention significantly enhances the SERS signal and improves the detection sensitivity by constructing a dendritic Pt nanowire structure.

[0058] Fluorescence background interference: In traditional SERS detection, background fluorescence signals often interfere with the detection results, resulting in reduced detection accuracy. The present invention successfully quenches the fluorescence background of TMV-Pt nanowires by introducing rGO, greatly improving the clarity and accuracy of SERS signals.

[0059] Complex structures are difficult to prepare: In the prior art, it is difficult to construct nanoprobes with complex structures, and the process is complex and unstable. The present invention simplifies the preparation process of complex nanostructures through the TMV template and Pt nanoparticle guided growth method, and can stably control the size and structure of the nanoprobe by finely regulating the reaction conditions.

[0060] 3. Significant technological progress

[0061] Significant improvement in sensitivity and selectivity: The present invention significantly improves the sensitivity and selectivity of the SERS probe by constructing dendritic Pt nanowires on the surface of TMV and combining the chemical enhancement effect of rGO. It can effectively detect trace amounts of pesticide residues in water with a lower detection limit and superior performance.

[0062] Elimination of background interference: Through the introduction of rGO, the fluorescence background of the nanoprobe was successfully quenched, effectively solving the signal interference problem in traditional SERS probe detection, thereby improving detection accuracy and data reliability.

[0063] Controllability of nanostructure: TMV is used as a biological template, combined with ascorbic acid (AA) and sodium polystyrene sulfonate (PSS) to finely control the growth direction and size of Pt nanowires, making the preparation of complex nanostructures simpler and more controllable, and the structural stability is high, which is suitable for actual water quality detection applications.

[0064] Green and environmentally friendly preparation method: Compared with the traditional chemical synthesis method, the present invention adopts TMV biological template, avoids the use of toxic chemical reagents, and the process is green and environmentally friendly, which meets the current requirements for sustainable development.

[0065] In summary, the present invention constructs a highly sensitive and highly selective SERS nanoprobe through the innovative TMV template-induced growth of platinum nanowires and their composite with rGO, which significantly improves the sensitivity and accuracy of pesticide residue detection in water and solves the problems of insufficient sensitivity, background interference and preparation complexity in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 It is a flow chart of a method for preparing a SERS nanoprobe for quantitative detection of pesticide residues in water provided by an embodiment of the present invention.

[0067] Figure 2 It is a schematic diagram of a detection method for a SERS nanoprobe for quantitative detection of pesticide residues in water provided in an embodiment of the present invention.

[0068] Figure 3 It is the surface protein capsid structure of TMV drawn by PyMOL provided in an embodiment of the present invention, and the blue area represents arginine residues (PDB file: 2TMV).

[0069] Figure 4 These are transmission electron micrographs provided in the embodiments of the present invention; (A) TMV (scale bar: 100 nm), (B) TMV-Pt seeds (scale bar: 200 nm) and (C) TMV-DPtNCs (scale bar: 100 nm).

[0070] Figure 5 3 is a particle size distribution diagram of TMV-Pt seeds (A) and TMV-DPtNCs (B) provided in the embodiments of the present invention.

[0071] Figure 6 It is the ultraviolet spectra of wild-type TMV and TMV-DPtNCs provided in the embodiments of the present invention.

[0072] Figure 7 is an X-ray diffraction (XRD) pattern of TMV-DPtNCs provided in an embodiment of the present invention, and the (111) and (200) peaks are typical diffraction peak patterns of the Pt fcc structure.

[0073] Figure 8 The Raman spectra of TMV and TMV-DPtNCs at three laser wavelengths ((A) 633 nm, (B) 785 nm, (C) 532 nm) provided in the embodiment of the present invention (the R6G addition concentration is 10 -5 M).

[0074] Fig. 9The Raman spectra of TMV-DPtNCs@rGO as the substrate provided by the embodiment of the present invention, wherein (A) the rGO concentration is 0.5 g / L, the R6G addition concentration is 10 -5 M, (B) rGO concentration is 0.005 g / L, R6G addition concentration is 10 -5 M.

[0075] Fig.10 The present invention provides a method for preparing TMV-DPtNCs@rGO-based R6G with different concentrations (10 -4 M to 10 -9 M) Raman spectrum.

[0076] Fig.11 The TMV-DPtNCs@rGO substrate (R6G is 10 -6 M) Raman spectrum obtained by performing 10 Raman scans at different positions (with an RSD value of 4.19%).

[0077] Fig.12 The present invention provides a method for preparing TMV-DPtNCs@rGO based on different concentrations of damanlin (10 -4 M to 10 -7 M) Raman spectrum (A) and intensity / concentration linear relationship diagram (B).

[0078] Fig.13 The present invention provides a method for preparing TMV-DPtNCs@rGO with different concentrations of triazophos (10 -4 M to 10 -7 M) Raman spectrum (A) and intensity / concentration linear relationship diagram (B).

[0079] Fig.14 The present invention provides a method for preparing TMV-DPtNCs@rGO with different concentrations of carbaryl (10 -4 M to 10 -7 M) Raman spectrum (A) and intensity / concentration linear relationship diagram (B).

[0080] Fig.15 This is a comparison chart of the Raman spectrum detection values ​​and actual added values ​​of three pesticide residues in lake water by TMV-DPtNCs@rGO provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0081] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0082] like Figure 1 As shown, a method for preparing a SERS nanoprobe for quantitative detection of pesticide residues in water provided by an embodiment of the present invention comprises the following steps:

[0083] Step 1: Using tobacco mosaic virus (TMV) as a biological template, using the widely distributed arginine residues on its surface as inducing groups, reducing metal ions platinum (Pt) (single or multiple metals such as gold, silver, palladium, platinum, copper, etc. can be selected) to promote the rapid nucleation of Pt atoms on the outer surface of TMV to form TMV-Pt seeds;

[0084] Step 2: Use sodium polystyrene sulfonate (PSS) and ascorbic acid (AA) to finely control the growth direction of Pt seeds on the TMV surface, and promote Pt to form dendritic clusters in a limited space (2.5 nm in the axial direction and 3.5 nm in the radial direction).

[0085] Step 3: The diameter of the dendritic metal clusters was adjusted by controlling the reaction time, and finally Pt-covered one-dimensional nanowires (TMV-DPtNCs) were formed on the TMV surface.

[0086] Step 4: Prepare TMV-DPtNCs@rGO composite material using reduced graphene oxide (rGO); the addition of rGO can quench the fluorescence background of TMV-DPtNCs and provide a chemical enhancement (CM) effect for surface enhanced Raman scattering (SERS).

[0087] The specific preparation process is as follows:

[0088] 1. Purification of TMV:

[0089] Take about 100g of tobacco leaves (frozen and stored in a -80℃ refrigerator), crush them by hand and add them to a juicer, add 300mL potassium phosphate buffer solution (100mmol / L, pH 7.8) and 1mL β-mercaptoethanol, crush them, and filter out the residue with double-layer gauze. Centrifuge the filtrate at 10580r / min for 20min at 4℃, with a rotor of Beckman JLA-16.250, and take the supernatant. Add an equal volume of chloroform and n-butanol mixed solvent (v:v=1:1) and stir in an ice bath for 30min. Centrifuge the mixture at 6045r / min for 10min at 4℃, with a rotor of Beckman JLA-16.250. Take the upper layer (water layer) and add PEG 8000 to a final concentration of 10% (w / V). Add sodium chloride to a final concentration of 200mmol / L and stir in an ice bath for 30min. The mixed solution was centrifuged at 14106rpm for 30min at 4℃, with a rotor of Beckman JLA-16.250. After carefully discarding the supernatant, 50mL of potassium phosphate buffer solution (10mmol / L, pH7.8) was added for redissolution, and centrifuged at 9500r / min for 15min, with a rotor of Beckman25.5, and the supernatant was taken. Centrifuged at 45000r / min for 2.5h at 4℃, with a rotor of Beckman50.2Ti. The precipitate was redissolved with 10mL of potassium phosphate buffer solution (10mmol / L, pH7.8). The heavy solution was centrifuged at 9500r / min for 15min at 4℃ and refrigerated at 4℃. The TMV concentration was semi-quantitatively determined by measuring the ultraviolet absorption at 260nm, and the extinction coefficient was 3.1cm 2 / g.

[0090] 2. Synthesis of TMV-Pt seeds

[0091] 15 mg of sodium polystyrene sulfonate (PSS) was added to a TMV aqueous solution (15 mL, 6 mg) and stirred to dissolve; 5 mL of K2PtCl4 solution (20 mM) was added dropwise and stirred at room temperature for 30 min. The arginine groups evenly distributed on the surface of TMV were used to adsorb [PtCl4] 2- Then, a freshly prepared NaBH4 solution (20 mL, 5 mM) was quickly poured in at once and the mixture was stirred rapidly for 2 h to generate Pt nanoparticles with a particle size of about 2 nm on the surface of TMV.

[0092] After the reaction is completed, the reaction solution is transferred to a centrifuge tube, and 20% glycerol is used as a cushion layer. Centrifuge at 9400g for 30min. Carefully remove the supernatant, add ultrapure water to the precipitate to resuspend, centrifuge at 6000g for 20min, and wash the precipitate. After washing three times, add ultrapure water to the precipitate to resuspend, centrifuge at 500g for 10min, collect the supernatant and store at 4°C, which is the TMV-Pt seed.

[0093] 3. Synthesis of continuous multichannel Pt nanowires with dendritic structure (TMV-DPtNCs)

[0094] Weigh 240 mg of polyvinylpyrrolidone (PVP) (K-30), dissolve it in 32 mL of water, add TMV-Pt seeds to make the final concentration of 10 μg / mL; add K2PtCl4 solution (4 mL, 10 mM) under stirring; finally, add freshly prepared ascorbic acid (AA) solution (4 mL, 100 mM) and stir at room temperature to react overnight.

[0095] After the reaction is completed, the reaction solution is transferred to a centrifuge tube, centrifuged at 6500g for 20 minutes, and the supernatant is discarded. Ultrapure water is added to the precipitate to resuspend it, and the precipitate is washed by centrifugation. After washing three times, the precipitate is dispersed in ultrapure water and stored at 4°C to obtain TMV-DPtNCs.

[0096] 4. Characterization of TMV-DPtNCs

[0097] TMV-DPtNCs were characterized by transmission electron microscopy, ultraviolet spectrometer, X-ray diffractometer, inductively coupled plasma mass spectrometer, etc. The results showed that TMV-DPtNCs were one-dimensional nanowires covered with dendritic Pt clusters.

[0098] (1) Scanning electron microscopy (TEM)

[0099] There are 2130 pairs of arginine residues outside the TMV. The distance between the two closest pairs of arginine residues along the axial direction is about 2.5nm, and the distance along the radial direction is about 3.5nm ( Figure 3 and Figure 4 (A)). Therefore, when [PtCl6] 2- When the ions are reduced, the arginine residues, which are strong ligands for Pt, induce the preferential nucleation of Pt atoms outside the TMV. At the same time, the hydrophobic part (polystyrene) can adsorb on the Pt surface, while the hydrophilic part (SO 3- ) carries a strong negative charge, providing electrostatic repulsion to avoid aggregation of Pt. Therefore, the dispersed TMV was tailored to evenly distribute TMV-Pt seeds ( Figure 4 (B)). Next, under the guidance of ascorbic acid (AA), the TMV exterior grew into dendritic Pt clusters, which increased in size and spread over the entire TMV surface over time ( Figure 4 (C)).

[0100] The particle size distribution of TMV seeds and TMV-DPtNCs is shown in Figure 5 As shown, the average particle size of TMV-Pt seeds is 18.9±1.0nm ( Figure 5(A)), the average particle size of TTMV-DPtNCs is 62.1±3.7nm ( Figure 5 (B)).

[0101] (2) UV spectra of TMV and TMV-DPtNCs

[0102] like Figure 6 As shown, TMV has a characteristic absorption peak at 260 nm, while TMV-DPtNCs covered with metallic Pt do not have this characteristic peak, indicating uniform full coverage of Pt.

[0103] (3) X-ray diffraction (XRD) results of TMV-DPtNCs

[0104] like Figure 7 As shown in the X-ray diffraction (XRD) spectrum, the (111) and (200) peaks are typical diffraction peaks of the Pt fcc structure. The XRD results show that the Pt nanostructure grown on the surface of TMV (Tobacco Mosaic Virus) maintains the typical fcc lattice structure of Pt.

[0105] 5. Raman activity of TMV-DPtNCs

[0106] (1) Sample preparation

[0107] Take 10 μL of TMV stock solution and TMV-DPtNCs and rhodamine 6G (R6G) (concentration of 10 -5 M) aqueous solution) 10 μL, mix well and set aside.

[0108] (2) Testing

[0109] Take 2 μL of the above solution, place it on a single concave slide, dry it, and then test it at 532 nm, 633 nm, and 785 nm.

[0110] like Figure 8 As shown, there is no characteristic peak at 633nm; the Raman shift at 785nm is 1368cm -1 There is a characteristic peak at 532nm, which is presumed to be TMV itself, and the characteristic peak of R6G was not measured; in addition to the characteristic peak of TMV at 532nm, a relatively significant Raman enhancement effect was observed in TMV-DPtNCs. However, since the virus sample itself has a high fluorescence, the entire spectrum has a high background. Therefore, it is necessary to reduce the fluorescence to improve the Raman effect.

[0111] VI. Preparation of TMV-DPtNCs@rGO Composite Stock Solution

[0112] 100 μL of TMV-DPtNCs and a certain amount of rGO were mixed to ensure that the final concentration of rGO was 0.005 g / L and stored in a refrigerator at -4 °C.

[0113] Combining carbon-based nanomaterials, such as graphene or its oxidized form (GO), with noble metal nanostructures is a synergistic approach to improving SERS performance. These hybrid materials enhance the adsorption of analytes through non-covalent interactions, including π-π stacking and electrostatic forces. The proximity of molecules to the graphene surface changes its electronic structure, thereby increasing polarizability and enhancing the Raman scattering cross section. This effect, along with dynamic charge transfer, contributes to the chemical mechanism (CM) of enhanced SERS. Another important advantage of adding graphene-based materials is their ability to suppress excess fluorescence from target analytes and background molecules. This fluorescence quenching effect can improve the signal-to-noise ratio, which is particularly beneficial for detecting trace pollutants or biomarkers in complex matrices. Therefore, the present invention uses a monolayer of rGO to reduce the fluorescence background of TMV-DPtNCs.

[0114] like Figure 2 As shown, a detection method of a SERS nanoprobe for quantitative detection of pesticide residues in water provided by an embodiment of the present invention comprises the following steps:

[0115] Step 1: Using the fluorescent probe molecule rhodamine 6G (R6G) in combination with Raman spectroscopy, the Raman enhancement effect of TMV-DPtNCs@rGO was evaluated and its enhancement factor (EF) was calculated;

[0116] Step 2, evaluate the SERS enhancement effect of TMV-DPtNCs@rGO on three commonly used pesticides (diflunisal, carbaryl, and triazophos); establish the linear relationship between the characteristic peak intensity and concentration of these pesticides by the least squares method for quantitative detection;

[0117] Step 3, the standard addition method was used to evaluate the detection ability of TMV-DPtNCs@rGO for three pesticide residues in actual lake water samples.

[0118] 1. Testing the Raman performance of TMV-DPtNCs@rGO composite for fluorescent probe (R6G)

[0119] Take 2 μL of TMV-DPtNCs@rGO composite and 2 μL of different concentrations (10 -4 M to 10 -9 The mixture was mixed with an aqueous solution of R6G (M), placed on a hydrophilic silicon wafer and dried, and then detected using a Raman spectrometer to calculate the EF value, RSD value, etc.

[0120] like Fig. 9 As shown in Figure 2, when the dosage of rGO is 0.5 g / L, the characteristic peak of R6G can be seen, but the characteristic peak of rGO is also obvious ((1361 cm -1 (D) belt and 1580cm -1When the rGO concentration was reduced to 0.005 g / L, the characteristic peak of rGO (1361 cm -1 (D) belt and 1580cm -1 (G band)) almost disappeared, and only the characteristic peak of the probe molecule R6G was shown.

[0121] The minimum detection limit of TMV-DPtNCs@rGO for R6G.

[0122] like Fig.10 As shown in the Raman spectra of R6G at different concentrations, it can be seen that when the concentration drops to 10 -9 The characteristic peaks of R6G are still clearly identifiable when the concentration of M is less than 1.5, and the characteristic peaks are assigned to (614cm-1δ(CCC), 774cm-1γ(CCC), 1363cm-1δ(CH), 1509cm-1δ(CH), and 1650cm-1δ(CH)).

[0123] Repeatability test of TMV-DPtNCs@rGO

[0124] like Fig.11 As shown, R6G in 10 -6 The test was carried out 10 times at different positions at a concentration of M, and the results are as follows: the RSD value is 4.19%, which shows that the TMV-DPtNCs@rGO substrate has good uniformity and repeatability.

[0125] Enhancement Factor (EF) Value

[0126] The enhancement factor (EF) is an important parameter in the field of SERS detection, which represents the amplification of the SERS signal of the interaction between the signal molecule and the nanostructure surface. The calculation of EF requires careful evaluation of the signal intensity and the number of molecules under SERS and normal Raman conditions, and is calculated according to the following formula.

[0127]

[0128] in:

[0129] I SERS and I Ref They represent the Raman spectrum intensity after SERS enhancement and the intensity during ordinary Raman testing, respectively.

[0130] C SERS and C Ref They represent the concentration during SERS detection and the concentration during ordinary Raman testing, respectively.

[0131] V SERS and V Ref They represent the volume during SERS detection and the volume during ordinary Raman testing, respectively.

[0132] S SERS and S Ref They represent the surface area formed by the solution, in the case of SERS and in the case of ordinary Raman testing, respectively.

[0133] According to the calculation formula of EF value, the result of using R6G as the probe molecule is EF=1.18×10 6

[0134] 2. Test the Raman performance of TMV-DPtNCs@rGO composite for three pesticides (triazophos, cypermethrin and carbaryl).

[0135] Take 2 μL of TMV-DPtNCs@rGO composite and 2 μL of different concentrations (10 -4 M to 10 -7 The mixture of pesticides was placed on a hydrophilic silicon wafer and dried before detection by Raman spectrometer. The least square method was used to establish the linear relationship between the characteristic peak intensity and concentration of the pesticide for quantitative detection.

[0136] (1) Dapoxetine

[0137] like Fig.12 As shown in (A), the minimum detection limit of TMV-DPtNCs@rGO for cypermethrin is 10 -7 M. The characteristic peaks of damanlin are assigned to 644cm-1(δ(ring)), 711cm-1(ν(CS); ν(CCC); ν(CC)), 935cm-1(ρ(CH3)), 79cm-1(ν(ring); δ(CH)), 1489cm-1(ν(CN);

[0138] 1489cm -1 The linear relationship between intensity and concentration was calculated by the characteristic peak intensity of chloranil. Fig.12 (B) It can be seen that it has good linearity and can be used for quantitative calculations.

[0139] (2) Triazophos

[0140] like Fig.13 As shown in Figure 2, the minimum detection limit of TMV-DPtNCs@rGO for triazophos is 10 -7 M. The characteristic peaks of triazophos are assigned to 959cm-1(δ(CNN),δ(ring)); 1002cm-1(ν(ring)); 1410cm-1(ν(CN); 1601cm-1(ν(C=C);

[0141] 1002cm -1 The linear relationship between intensity and concentration is calculated based on the characteristic peak intensity at Fig.13(B) It can be seen that it has good linearity and can be used for quantitative calculations.

[0142] (3) Carbaryl

[0143] like Fig.14 As shown in Figure 2, the minimum detection limit of TMV-DPtNCs@rGO for triazophos is 10 -7 M. The characteristic peaks of carbaryl are assigned to 1440cm-1(δ(CH)); 1514cm-1(δ(NH),δ(CH)m); 1572cm-1(Ring deformation).

[0144] 1440cm -1 The linear relationship between intensity and concentration is calculated using the characteristic peak intensity at .

[0145] 3. Detection of three pesticides in real environmental water samples using TMV-DPtNCs@rGO composites

[0146] Take natural lake water and add three pesticides of different concentrations (5.00×10 -4 M to 5.00×10 -7 M), and then filtered using a 0.22 μm nylon filter membrane. Then, 2 μL of water sample was mixed with 2 μL of TMV-DPtNCs@rGO composite, placed on a hydrophilic silicon wafer for drying and then subjected to Raman detection.

[0147] The detection conditions of the above Raman spectrometer are: 532 nm, 1% energy, 5 seconds integration time, and integration times: 1.

[0148] Raman test results of TMV-DPtNCs@rGO on three pesticide residues in lake water Fig.15 shown.

[0149] The local lake water in Weifang, Shandong was added with three pesticides at different concentrations, and then filtered through a 0.22μm nylon membrane for Raman testing. Each concentration was tested 6 times, and the results were averaged.

[0150] The results in Table 1 show that even when the pesticide concentration is as low as When the concentration is lowered, the recovery rates of the three pesticide residues are between 80-120%, which can meet the detection requirements of real samples. However, when the concentration is further reduced, the difference between the test results and the actual values ​​is large.

[0151] Table 1 Average recoveries and RSDs of three pesticides in spiked water samples (n=6)

[0152]

[0153]

[0154] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A SERS nanoprobe for quantitative detection of pesticide residues in water, characterized in that: The following steps are involved: Step 1: Using tobacco mosaic virus (TMV) as a biological template, using the widely distributed arginine residues on its surface as inducing groups, reducing the metal ion platinum Pt, prompting Pt atoms to rapidly nucleate on the outer surface of TMV to form TMV-Pt seeds; Step 2: Use sodium polystyrene sulfonate (PSS) and ascorbic acid (AA) to finely control the growth direction of Pt seeds on the TMV surface, and promote Pt to form a dendritic structure cluster in a limited space of 2.5 nm in the axial direction and 3.5 nm in the radial direction. Step 3: By controlling the reaction time, the diameter of the dendritic metal clusters is adjusted, and finally Pt-covered one-dimensional nanowires TMV-DPtNCs are formed on the TMV surface; Step 4, preparing TMV-DPtNCs@rGO composite material by reducing graphene oxide rGO; The addition of rGO quenched the fluorescence background of TMV-DPtNCs and provided a chemical enhancement (CM) effect for surface enhanced Raman scattering (SERS).

2. The SERS nanoprobe for quantitative detection of pesticide residues in water according to claim 1, characterized in that: The purification method of tobacco mosaic virus (TMV) includes: Take about 100g of tobacco leaves, crush them, add 300mL potassium phosphate buffer solution and 1mL β-mercaptoethanol, crush them, and filter out the residue with double-layer gauze; centrifuge the filtrate at 10580r / min for 20min at 4°C, and take the supernatant; add an equal volume of chloroform-n-butanol mixed solvent, and stir in an ice bath for 30min; centrifuge the mixture at 6045r / min for 10min at 4°C, and take the upper layer; add PEG8000 to a final concentration of 10% (w / V); add sodium chloride to a final concentration of 200mmol / L, and stir in an ice bath for 30min; centrifuge the mixture at 14106rpm for 30min at 4°C, pour out the supernatant, add 50mL potassium phosphate buffer solution (10mmol / L, pH 7.8) Redissolve, centrifuge at 9500 r / min for 15 min, take the supernatant; centrifuge at 45000 r / min for 2.5 h at 4°C, and redissolve the precipitate with 10 mL potassium phosphate buffer solution; centrifuge the resolubilized solution at 9500 r / min for 15 min at 4°C, and store at 4°C; the TMV concentration is semi-quantitatively determined by measuring the ultraviolet absorption at 260 nm, and the extinction coefficient is 3.1 cm 2 / g.

3. The SERS nanoprobe for quantitative detection of pesticide residues in water according to claim 1, characterized in that: Synthesis method of TMV-Pt seeds: 15 mg of sodium polystyrene sulfonate (PSS) was added to the TMV aqueous solution and stirred to dissolve; 5 mL of K2PtCl4 solution was added dropwise and stirred at room temperature for 30 min to absorb [PtCl4] using the arginine groups evenly distributed on the surface of TMV. 2- Then, the freshly prepared NaBH4 solution was quickly poured in at once and the mixture was stirred rapidly for 2 h to generate Pt nanoparticles with a particle size of about 2 nm on the surface of TMV. After the reaction is completed, the reaction solution is transferred to a centrifuge tube, cushioned with 20% glycerol, and centrifuged at 9400g for 30 minutes; carefully remove the supernatant, add ultrapure water to the precipitate to resuspend, centrifuge at 6000g for 20 minutes, and wash the precipitate; after washing three times, add ultrapure water to the precipitate to resuspend, centrifuge at 500g for 10 minutes, collect the supernatant and store it at 4°C, which is the TMV-Pt seed.

4. The SERS nanoprobe for quantitative detection of pesticide residues in water according to claim 1, characterized in that: Synthesis method of continuous multi-channel Pt nanowires with dendritic structure: Weigh 240 mg of polyvinylpyrrolidone (PVP) (K-30), dissolve it in 32 mL of water, add TMV-Pt seeds to make the final concentration 10 μg / mL; add K2PtCl4 solution under stirring; finally add freshly prepared ascorbic acid (AA) solution, stir and react at room temperature overnight; After the reaction is completed, the reaction solution is transferred to a centrifuge tube, centrifuged at 6500g for 20 min, and the supernatant is discarded; ultrapure water is added to the precipitate to resuspend it, and the precipitate is washed by centrifugation; After washing three times, the precipitate was dispersed in ultrapure water and stored at 4°C to obtain TMV-DPtNCs.

5. The SERS nanoprobe for quantitative detection of pesticide residues in water according to claim 1, characterized in that: Preparation method of TMV-DPtNCs@rGO composite stock solution: 100 μL TMV-DPtNCs and a certain amount of rGO were mixed to ensure that the final concentration of rGO was 0.005 g / L and stored in a refrigerator at -4 °C.

6. A detection method for quantitative detection of pesticide residues in water using a SERS nanoprobe as claimed in any one of claims 1 to 5, characterized in that: The following steps are involved: Step 1: Using the fluorescent probe molecule rhodamine 6G (R6G) in combination with Raman spectroscopy, the Raman enhancement effect of TMV-DPtNCs@rGO was evaluated and its enhancement factor (EF) was calculated; Step 2: evaluate the SERS enhancement effect of TMV-DPtNCs@rGO on three commonly used pesticides: cypermethrin, carbaryl, and triazophos; establish the linear relationship between the characteristic peak intensity and concentration of these three pesticides by the least squares method for quantitative detection; Step 3, the standard addition method was used to evaluate the detection ability of TMV-DPtNCs@rGO for three pesticide residues in actual lake water samples.

7. The detection method of the SERS nanoprobe for quantitative detection of pesticide residues in water according to claim 6, characterized in that: Testing the Raman performance of TMV-DPtNCs@rGO composite for fluorescent probe (R6G): 2 μL of TMV-DPtNCs@rGO complex was mixed with 2 μL of R6G aqueous solution of different concentrations, placed on a hydrophilic silicon wafer and dried, and then detected using a Raman spectrometer to calculate the EF value, RSD value, etc.

8. The detection method of SERS nanoprobe for quantitative detection of pesticide residues in water according to claim 6, characterized in that: The Raman performance of TMV-DPtNCs@rGO composite for triazophos, cypermethrin and carbaryl was tested: 2 μL of TMV-DPtNCs@rGO complex was mixed with 2 μL of pesticides of different concentrations, placed on a hydrophilic silicon wafer and dried, and then detected using a Raman spectrometer. The least squares method was used to establish a linear relationship between the characteristic peak intensity and concentration of the pesticide for quantitative detection.

9. The detection method of SERS nanoprobe for quantitative detection of pesticide residues in water according to claim 6, characterized in that: The TMV-DPtNCs@rGO composite was used to detect three pesticides in real environmental water samples: Take lake water from a natural environment, add three pesticides of different concentrations, and then filter it using a 0.22μm nylon filter membrane; then take 2μL of the water sample and mix it with 2μL of the TMV-DPtNCs@rGO complex, place it on a hydrophilic silicon wafer, dry it, and then perform Raman detection.

10. The detection method of SERS nanoprobe for quantitative detection of pesticide residues in water according to claim 6, characterized in that: The detection conditions of the Raman spectrometer are: 532 nm, 1% energy, 5 seconds integration time, and integration times: 1.

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